Genetically engineered microorganisms and processes for the production of cannabinoids from a carbon source precursor

Abstract

A method is provided for biosynthetic production of cannabinoids in microorganisms from a carbon source precursor. This method describes the genetic modifications needed to engineer microorganisms to produce cannabinoids as well as a method for identifying and quantifying cannabinoids from fermentation broth. A system is also provided for tuning the method to produce different cannabinoids of interest by systematically modulating the enzymes encoded by the genetic modifications introduced in the microorganism.

What is claimed is: 
     
       1. A genetically modified microorganism that produces at least one cannabinoid from a carbon source precursor, wherein the microorganism is  E. coli  comprising a recombinantly incorporated mutated FadD gene having a nucleotide sequence of SEQ ID NO: 10 at the genomic location of a FadE gene of the microorganism to express the mutated FadD enzyme and simultaneously knock out the FadE gene of the microorganism. 
     
     
       2. The genetically modified microorganism according to  claim 1 , wherein the microorganism is transformed with a plasmid to express a polypeptide encoding olivetol synthase having a first amino acid sequence comprising the amino acid sequence of SEQ ID NO. 2, olivetolic acid cyclase having a second amino acid sequence comprising the amino acid sequence of SEQ ID NO. 4, and CsPT1 having a third amino acid sequence comprising the amino acid sequence of SEQ ID NO. 6. 
     
     
       3. The genetically modified microorganism according to  claim 2 , wherein the plasmid comprises a nucleotide sequence having the nucleotide sequence of SEQ ID NO: 9. 
     
     
       4. The genetically modified microorganism according to  claim 1 , wherein the microorganism is transformed with a plasmid comprising a nucleotide sequence of SEQ ID NO: 11. 
     
     
       5. The genetically modified microorganism according to  claim 1 , further comprising recombineered metabolic protein degradation valves for one or more of a pgi gene, a zwf gene and a gltA gene by adding a C-terminal DAS+4 tag having the nucleotide sequence SEQ ID NO: 12 to the respective one or more of the pgi gene, the zwf gene and the gltA gene. 
     
     
       6. A plasmid that encodes olivetol synthase having an amino acid sequence of SEQ ID NO: 2, olivetolic acid cyclase having an amino acid sequence of SEQ ID NO: 4, CsPT1 having an amino acid sequence of SEQ ID NO: 6, and a Glutathione-S-Transferase solubility tag having an amino acid sequence of SEQ ID NO: 8. 
     
     
       7. A Vector comprising the plasmid of  claim 6 . 
     
     
       8. The genetically modified microorganism according to  claim 2 , wherein the plasmid further comprises nucleotide sequences encoding a Glutathione-S-Transferase (GST) solubility tag with a thrombin cleavage site having the amino acid sequence of SEQ ID NO: 8 in the same open reading frame as CsPT1. 
     
     
       9. The genetically modified microorganism according to  claim 1 , further comprising: a first nucleotide sequence of SEQ ID NO: 9; and a second nucleotide sequence of SEQ ID NO: 11. 
     
     
       10. A genetically modified microorganism that produces at least one cannabinoid from a carbon source precursor, wherein the microorganism is  S. cerevisiae  and has been transformed with a plasmid to express a polypeptide encoding olivetol synthase having the amino acid sequence of SEQ ID NO: 2, a polypeptide encoding olivetolic acid cyclase having the amino acid sequence of SEQ ID NO: 4, a polypeptide encoding CsPT1 having the amino acid sequence of SEQ ID NO: 6, and further comprising a Glutathione-S-Transferase (GST) solubility tag with a thrombin cleavage site having the amino acid sequence of SEQ ID NO: 8 in the same open reading frame as CsPT1. 
     
     
       11. The genetically modified microorganism according to  claim 10 , further comprising recombineered metabolic protein degradation valves for one or more of a pgi gene, a zwf gene and a gltA gene by adding a C-terminal DAS+4 tag having the nucleotide sequence SEQ ID NO: 12 to the respective one or more of the pgi gene, the zwf gene and the gltA gene. 
     
     
       12. The plasmid of  claim 6  comprising the nucleotide sequence of SEQ ID NO: 9. 
     
     
       13. A Vector comprising the plasmid of  claim 12 .